Blostein R, Daly S E, Boxenbaum N, Lane L K, Arguello J M, Lingrel J B, Karlish S J, Caplan M J, Dunbar L
McGill Univ., Montreal, Canada.
Acta Physiol Scand Suppl. 1998 Aug;643:275-81.
This paper summarizes experiments concerned with the functional consequences of mutations in cytoplasmic regions of the alpha 1 subunit of the Na,K-ATPase, in particular the amino terminus, the first cytoplasmic loop between transmembrane segments M2 and M3, and the major cytoplasmic loop between M4 and M5. In the first mutation (alpha 1M32), 32 residues were removed from the N-terminus. The second mutation (E233K) was in the putative beta strand of M2-M3 loop and the third, comprised the replacement of the amino terminal half of loop M4-M5 of the Na,K-ATPase with the homologous segment (residues 356-519) of the gastric H,K-ATPase. The first two mutations, either separately or in combination (alpha 1M32E233K), shift the equilibrium between the major conformational states of the enzyme, E1 and E2, in favor of E1 as manifested by increased apparent affinity for ATP, lower catalytic turnover, and decreased sensitivity to inhibition by vanadate. The striking changes observed with alpha 1M32E233K suggests interactions between the N-terminus, the beta-strand in the M2-M3 loop and the catalytic phosphorylation site. The behavior of these mutants contrasts with that of least one mutant involving substitution of a residue in the putative cation binding pocket, namely S775A in the fifth transmembrane segment (Arguello, J.M., & Lingrel, J. B. J. Biol. Chem. 270: 22764-22771, 1995). Although its K+/ATP antagonism resembles that of the foregoing cytoplasmic mutants, its vanadate sensitivity is unaltered suggesting that changes in apparent affinity for ATP are secondary to changes in K+ ligation. The question of cation selectivity, in particular that of Na+ versus protons, has been addressed in structure/function analysis of a cytoplasmic chimera involving the M4-M5 loop. Transport studies performed in the presence or absence of Na+ and at low versus high pH indicate a marked alteration in cation affinity and/or selectivity. This results suggests coupling of an alteration in the large M4-M5 cytoplasmic domain to cation binding in, presumably, the juxtapositioned transmembrane domain.
本文总结了有关钠钾ATP酶α1亚基胞质区域突变功能后果的实验，特别是氨基末端、跨膜片段M2和M3之间的第一个胞质环以及M4和M5之间的主要胞质环。在第一个突变（α1M32）中，从N末端去除了32个残基。第二个突变（E233K）位于M2-M3环的假定β链中，第三个突变包括用胃H,K-ATP酶的同源片段（残基356-519）替换钠钾ATP酶M4-M5环的氨基末端一半。前两个突变单独或组合（α1M32E233K）都会改变酶的主要构象状态E1和E2之间的平衡，有利于E1，表现为对ATP的表观亲和力增加、催化周转率降低以及对钒酸盐抑制的敏感性降低。α1M32E233K观察到的显著变化表明N末端、M2-M3环中的β链与催化磷酸化位点之间存在相互作用。这些突变体的行为与至少一个涉及假定阳离子结合口袋中残基取代的突变体相反，即第五个跨膜片段中的S775A（Arguello，J.M.和Lingrel，J.B.《生物化学杂志》270:22764-22771，1995）。尽管其K+/ATP拮抗作用与上述胞质突变体相似，但其对钒酸盐的敏感性未改变，这表明对ATP的表观亲和力变化是K+结合变化的次要结果。在涉及M4-M5环的胞质嵌合体的结构/功能分析中，已经探讨了阳离子选择性问题，特别是Na+与质子的选择性问题。在有或没有Na+以及低pH与高pH条件下进行的转运研究表明阳离子亲和力和/或选择性有显著改变。这一结果表明，大的M4-M5胞质结构域的改变与阳离子结合相关，推测是在相邻的跨膜结构域中。
